Benjamin R. G. Johnson

Expanding 3D geometry for enhanced on-chip microbubble production and single step formation of liposome modified microbubbles

Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for ultra-sound (US) imaging and increasingly as delivery vehicles for targeted, triggered, therapeutic delivery. Microfluidics provides a reproducible means for microbubble production and surface functionalisation. In this study, microbubbles are generated on chip using flow-focussing microfluidic devices that combine streams of gas and liquid through a nozzle a few microns wide and then subjecting the two phases to a downstream pressure drop. While microfluidics has successfully demonstrated the generation of monodisperse bubble populations, these approaches inherently produce low bubble counts. We introduce a new micro-spray flow regime that generates consistently high bubble concentrations that are more clinically relevant compared to traditional monodisperse bubble populations. Final bubble concentrations produced by the micro-spray regime were up to 10(10) bubbles mL(-1). The technique is shown to be highly reproducible and by using multiplexed chip arrays, the time taken to produce one millilitre of sample containing 10(10) bubbles mL(-1) was ∼10 min. Further, we also demonstrate that it is possible to attach liposomes, loaded with quantum dots (QDs) or fluorescein, in a single step during MBs formation.

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimers disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.

Native E. coli inner membrane incorporation in solid-supported lipid bilayer membranes

Solid-supported bilayer lipid membranes (SBLMs) containing membrane protein have been generated through a simple lipid dilution technique. SBLM formation from mixtures of native Escherichia coli bacterial inner membrane (IM) vesicles diluted with egg phosphatidylcholine (egg PC) vesicles has been explored with dissipation enhanced quartz crystal microbalance (QCM-D), atomic force microscopy (AFM), attenuated total internal-reflection Fourier-transform infrared spectroscopy (ATR-FTIR), and fluorescence recovery after photobleaching (FRAP). QCM-D studies reveal that SBLM formation from vesicle mixtures ranging between 0% and 100% IM can be divided into two regimes. Samples with ≤40% IM form SBLMs, while samples of greater IM fractions are dominated by vesicle adsorption. FRAP experiments showed that the bilayers formed from mixed vesicles with ≤40% IM were fluid, and comprised a mixture of both egg PC and IM. ATR-FTIR measurements on SBLMs membranes formed with 30% IM confirm that protein is present. SBLM formation was also explored as a function of temperature by QCM-D and FRAP. For samples of 30% IM, QCM-D data show a decreased mass and viscoelasticity at elevated temperatures, and an increased fluidity is observed by FRAP measurements. These results suggest improved biomimetic characteristics can be obtained by forming and maintaining the system at, or close to, 37 °C.

Minimal F-Actin Cytoskeletal System for Planar Supported Phospholipid Bilayers

Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.

Effect of the structure of cholesterol-based tethered bilayer lipid membranes on ionophore activity.

Tethered bilayer lipid membranes (tBLM) are formed on 1) pure tether lipid triethyleneoxythiol cholesterol (EO(3)C) or on 2) mixed self-assembled monolayers (SAMs) of EO(3)C and 6-mercaptohexanol (6MH). While EO(3)C is required to form a tBLM with high resistivity, 6MH dilutes the cholesterol content in the lower leaflet of the bilayer forming ionic reservoirs required for submembrane hydration. Here we show that these ionic reservoirs are required for ion transport through gramicidin or valinomycin, most likely due to the thermodynamic requirements of ions to be solvated once transported through the membrane. Unexpectedly, electrochemical impedance spectroscopy (EIS) shows an increase of capacitance upon addition of gramicidin, while addition of valinomycin decreases the membrane resistance in the presence of K(+) ions. We hypothesise that this is due to previously reported phase separation of EO(3)C and 6MH on the surface. This results in ionic reservoirs on the nanometre scale, which are not fully accounted for by the equivalent circuits used to describe the system.

A Self-assembly Route for Double Bilayer Lipid Membrane Formation

Solid supported lipid membranes provide a simple biomimetic model system that is suitable for studying a wide range of membrane related phenomena and importantly permits the use of a number of surface analytical techniques from AFM to impedance spectroscopy to be used in characterising such processes. Additionally, it is also believed that such systems could find application for drug screening, biosensing or in protein separation/crystallisation. In nature there are however a number of situations in which double bilayers naturally occur, for example in mitochondria or complexes which span two lipid bilayers at gap junctions, and for this reason it would be desirable to be able to create double bilayer mimics as an extension of the supported bilayer field. It has been previously demonstrated that one can create such structures using the Langmuir–Blodgett technique, however, this has the drawback that it is not compatible with the incorporation of transmembrane proteins and the film is created by transfer through the air–water interface. Hence approaches based on self-assembly from vesicles would provide a significant advance in the type of applications that double bilayers could be used for. Murray et al. have recently demonstrated, a previously observed phenomenon, that a second bilayer can be assembled on top of a streptavidin protein film, attached to a first bilayer. Further, Chung et al. have also shown that giant unilamellar vesicle (GUV) rupture can lead to the formation of a second bilayer tethered to a first bilayer using complementary DNA sequences. Similarly, GUV rupture onto a lipid bilayer to form model intermembrane junctions has been reported by Kaizuka and Groves; and Tabaei et al. have demonstrated a method whereby DNA duplexes were used to tether multiple diskshaped lipid “bicelles” to a bilayer. While these systems could not be used for studying double bilayer phenomena directly, they demonstrate that the principle of achieving double bilayers via self-assembly is feasible. Here we describe a new method to form double bilayer lipid membranes (dBLMs) on solid supports using NHS/EDC chemistry [hydroxy-2,5-dioxopyrrolidine-3-sulfonicacid sodium salt (NHS) and N-Ethyl-N’-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) Figure 1]. In this approach, two biomembranes, one of which contains the amine-functionalised cholesterol derivative 1 and the other which contains the CO2H-funtionalised cholesterol derivative 2 are covalently joined together by an NHS/EDC mediated reaction 19] as shown schematically in Figure 1. Details of the synthesis and characterization of the cholesterol derivatives 1 and 2 are given in the Supporting Information. These dBLMs can be formed both on functionalized Au and on silicon oxide surfaces. Here we describe their formation on a silicon oxide surfaces, monitored using a combination of fluorescence microscopy and atomic force spectroscopy. Vesicles containing Egg PC, reagent 2, DOTAP, and the red fluorescently labeled lipid TR DHPE (molar ratio 63.5:18:18:0.5) were used to form a single supported bilayer on a glass cover slip in the usual way. As shown in Figure 2 a, the fluorescence of the TR DHPE within this first bilayer is clearly seen, using a Texas red filter, and is uniform, whilst an image of the same region using a FITC filter does not show any fluorescence (Figure 2 b). The lateral diffusion coefficient of the TR DHPE was obtained from fluorescence recovery after photobleaching (FRAP). The average value of D from three different samples was 1.3 0.2 mm s 1 with the mobile fraction of ~93 %. The COOH groups of the reagent 2 within this first sBLM were activated by incubating for 15 min with NHS/EDC (5:1 ratio) solution. After thoroughly rinsing with Milli-Q water to remove the excess NHS/EDC, vesicles containing Egg PC, reagent 1 and green fluorescent lipid D291(molar ratio 71:18:1) were added. A reaction occurred between reagents 1 and 2 and a second bilayer was formed, covalently linked through amide bonds to the first bilayer. After rinsing, the double bilayer was investigated by fluorescence microscopy. Figure 2 c, taken with the Texas red filter, indicates the fluorescence of the inner bilayer, while Figure 2 d, taken with the FITC filter, shows the fluorescence of the outer bilayer. It is likely that a small amount of lipid exchange occurs between the first (Texas Red-labelled) bilayer and the second set of vesicles (D291), and as such the fluorescence images alone are insufficient to confirm that a second bilayer is present. In order to confirm that a second bilayer was indeed formed on top of the first bilayer, AFM force-distance measurements were conducted at each stage of the dBLM preparation, as shown in Figure 2 e and f. In the case of the first lipid bilayer (Figure 2 e) it is evident that, as the tip compresses and subsequently penetrates the membrane, it ‘jumps’ a distance equivalent to a single bilayer plus water layer (approximately 5 nm) before making contact with the underlying support. In the case of the dBLM the force–distance curves (Figure 2 f) show two clear ‘jumps’, as the tip penetrates first the outer and then the inner [a] Dr. X. Han, M. R. Cheetham, Dr. S. D. A. Connell, Dr. B. R. G. Johnson, Prof. S. D. Evans School of Physics and Astronomy University of Leeds (UK) Fax: + (44) 113 343 1884 E-mail : s.d.evans@leeds.ac.uk [b] Dr. A. S. Achalkumar, Prof. R. J. Bushby Self-Organising Molecular Systems (SOMS) Centre University of Leeds (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.200900798.

Universal synthesis method for mixed phase TiO2(B)/anatase TiO2 thin films on substrates via a modified low pressure chemical vapour deposition (LPCVD) route

A universal method for the synthesis of mixed phase TiO2 bronze (B)/anatase titania thin films by Low Pressure Chemical Vapour Deposition (LPCVD) onto any substrate is presented. General LPCVD conditions were titanium isopropoxide (TTIP) and N2 gas as the precursor and carrier gas respectively, 600 °C nominal reaction temperature, and 15 min reaction time; a range of different substrates were investigated including: a silicon wafer, fused quartz, highly ordered pyrolytic graphite (HOPG) and pressed graphite flake (grafoil). X-ray diffraction, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning and transmission electron microscopy were used to characterise the thin films which exhibited a columnar morphology together with smaller equi-axed particles. Pre-treatment of substrates by spraying with a Na-containing solution was found to encourage the crystallization of TiO2(B) during the LPCVD process. Increasing the concentration of Na in the pre-treatment process resulted in a higher proportion of TiO2(B) in the thin films up to an optimum condition of 0.75% w/v of Na. Na diffusion from the substrate surface into the adjacent TiO2 is the proposed mechanism for promoting TiO2(B) formation as opposed to the anatase phase with Density Functional Theory (DFT) modelling suggesting the presence of Na stabilises the TiO2(B) phase. Dye degradation tests indicate an increased photocatalytic activity for mixed phase anatase/TiO2(B) thin films.

Force spectroscopy of streptavidin conjugated lipid coated microbubbles

AbstractForce microscopy has been used to investigate the mechanical properties of phospholipid coated microbubbles and to quantify their stiffness. The mechanical properties were investigated using tipless cantilevers to compress microbubbles attached to a gold surface under aqueous conditions. The phospholipid microbubbles were produced by microfluidic flow focusing and were found to have stiffness of 25 mN m–1. The attachment of a streptavidin coating increased the microbubble stiffness by a factor of 30 to ∼750 mN m–1. Further, estimation of the frequency response based on values of stiffness obtained by force spectroscopy seems reasonable in comparison with those of an uncoated bubble and a polyethylene glycol coated Bracco SonoVue BR14 bubble, suggesting that the present approach may provide useful information for the development of novel microbubble coatings.

Actin Assembly at Model-Supported Lipid Bilayers

We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant-making this a practical approach for creating stable lipid-actin systems via self-assembly.

Oxidation of tertiary amine-derivatized surfaces to control protein adhesion.

Selective oxidation of ω-tertiary amine self-assembled thiol monolayers to tertiary amine N-oxides is shown to transform the adhesion of model proteins lysozyme and fibrinogen upon them. Efficient preparation of both secondary and tertiary linker amides as judged by X-ray photoelectron spectroscopy (XPS) and water droplet contact angle was achieved with an improved amide bond formation on gold quartz crystal microbalance (QCM) sensors using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate methanaminium uronium (HATU). Oxidation with hydrogen peroxide was similarly assessed, and adhesion of lysozyme and fibrinogen from phosphate buffered saline was then assayed by QCM and imaged by AFM. Tertiary amine-functionalized sensors adsorbed multilayers of aggregated lysozyme, whereas tertiary amine N-oxides and triethylene glycol-terminated monolayers are consistent with small protein aggregates. The surface containing a dimethylamine N-oxide headgroup and ethyl secondary amide linker showed the largest difference in adsorption of both proteins. Oxidation of tertiary amine decorated surfaces therefore holds the potential for selective deposition of proteins and cells through masking and other patterning techniques.